As an important component of blood, platelets are responsible for hemostasis in response to vascular injury and involved in the repairment of injured blood vessels. Low level of blood platelets can be life-threatening as it is prone to a mass loss of blood. At the present, platelet transfusion is a top choice for treatment for patients of thrombocytopenia. However, like other blood products, the platelets are short in shelf life, and are easy to be contaminated with blood pathogens such as hepatitis B virus and AIDS virus, and often elicit allergenic reactions among recipients.
Thrombopoietin (TPO) plays its role of growth factor for thrombopoiesis by binding to its receptor MPL, which is made up of three parts, MPL-EC(26-491aa) (extracellular domain), transmembrane domain, and cytoplasmic domain. In 1994, five groups of scientists simultaneously cloned TPO. The successful cloning of TPO had injected new hopes and approaches for the treatment of thrombocytopenia. However, the clinical data indicated that the efficacy of TPO towards thrombocytopenia varied among patients. At the same time, the side effects of TPO were also observed. Two of the major side effects were: [1] TPO activated platelets, stimulated its aggregation, and thus lead to the formation of blood clotting; and [2] antibodies to TPO being generated after TPO administration (Archimbaud E, et al. Blood. 1999; 94:3694-3701; Schiffer CA, et al, Blood. 2000; 95:2530-2535; Nash R, et al. Blood. 1997; 90 suppl. 1:262a). The search for alternative therapeutic proteins or cytokines continues in the art.
The yeast two-hybrid system is a suitable system for the study of protein-protein interactions. The system, which contains two expression plasmids (plasmid A and plasmid B) as well as a yeast host, takes advantage of the fact that yeast transcription factors, such as LEXA or Ga14, comprises two separate domains: DNA-binding domain (DNA-BD) and transcription activating domain (AD). Plasmid A expresses a fusion protein of a bait protein and the DNA-BD; and Plasmid B expresses a fusion protein of a protein of interest and AD. After co-transformation of the two plasmids into the yeast host, the interaction of the bait protein and the protein of interest brings the DNA-BD and AD into close contact, which activates the transcription of the reporter gene. Therefore the system can be used to isolate ligands of bait proteins. The kits Matchmaker Two-Hybrid System 3, Matchmaker LexA Two-Hybrid System (Clontech) are commercially-available examples of the system.